Syndecans and angiogenesis

ABSTRACT

The invention provides methods and materials related to modulating syndecan levels and angiogenesis in an animal. The invention provides syndecan polypeptides and nucleic acids encoding syndecan polypeptides, including dominant negative syndecan polypeptides. The invention also provides polynucleotides and polynucleotide analogues for modulating angiogenesis, as well as cells and embryos containing the polynucleotides and polynucleotide analogues. The invention further provides methods for identifying syndecan- and angiogenesis-modulating agents.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation in part of U.S. Ser. No. 10/347,022,filed Jan. 17, 2003, which claims priority from U.S. ProvisionalApplication Ser. No. 60/349,939, filed Jan. 18, 2002.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funding for the work described herein was provided in part by theNational Institutes of Health, grant numbers GM55877 and GM63904. Thefederal government may have certain rights in the invention.

TECHNICAL FIELD

The invention relates to methods and materials for modulatingangiogenesis in an animal by modulating the expression or activity ofsyndecan-2.

BACKGROUND

Proteoglycans are widely distributed, membrane-anchored glycoproteinsthat have covalently linked extracellular side-chains containingglycosaminoglycan (GAG) molecules such as heparan sulfate, a polymer ofrepeating disaccharide subunits. GAG side chains can be of differentlengths and are subject to modification by sulfation and epimerization,and their structures serve as specific recognition sites for variousligands, including growth factors, extracellular matrix components, andother cell surface molecules. Heparan sulfate proteoglycans have beenimplicated in the regulation of numerous cellular processes, includingcoagulation cascades, growth factor signaling, lipase binding andactivity, cell adhesion to the extracellular matrix and subsequentcytoskeletal organization, proliferation, differentiation, inflammation,microbial invasion, and tumor metastasis.

The syndecans make up a class of the heparan sulfate proteoglycans thatare present on most cell types. Syndecans appear to play modulatoryroles as coreceptors by presenting growth factors to their primaryreceptors or by increasing the infectivity of viruses by interactingwith their primary receptors. See, Woods (2001) J. Clin. Invest.107:935-941; and Elenius and Jalkanen (1994) J. Cell Sci. 107:2975-2982.Syndecans also have been implicated in neurite outgrowth, limbdevelopment, cell adhesion, and epithelial morphogenesis.

SUMMARY

The invention is based on the cloning of the zebrafish syndecan-2 gene(ec2) and the discovery that the encoded protein, EC2, is involved invasculogenesis and angiogenesis. This discovery indicates thatmodulation of syndecan levels would be useful for treating clinicalconditions associated with excessive or impaired angiogenesis andvasculogenesis. The invention therefore features materials and methodsfor modulating angiogenesis and vasculogenesis by modulating theexpression or function of syndecan polypeptides.

In one aspect, the invention features a method for inhibitingangiogenesis in a vertebrate. The method can include administering tothe vertebrate an effective amount of a cytoplasmically truncatedSyndecan-2 polypeptide. The truncated Syndecan-2 polypeptide can be adominant negative Syndecan-2 polypeptide (e.g., a polypeptide containingamino acids 1 to 193 of the sequence set forth in SEQ ID NO:2).

In another aspect, the invention features a method for inhibitingangiogenesis in a vertebrate. The method can include administering tothe vertebrate an effective amount of a nucleic acid containing asequence that encodes a cytoplasmically truncated Syndecan-2polypeptide. The construct can be expressed in the vertebrate to producethe truncated Syndecan-2 polypeptide. The truncated Syndecan-2polypeptide can be a dominant negative Syndecan-2 polypeptide (e.g., apolypeptide containing amino acids 1 to 193 of the sequence set forth inSEQ ID NO:2).

In another aspect, the invention features a method for killing a tumorcell. The method can include contacting the tumor cell with acytoplasmically truncated Syndecan-2 polypeptide. The contacting caninclude administering to the tumor cell a nucleic acid containing asequence that encodes the cytoplasmically truncated Syndecan-2polypeptide. The construct can be expressed in the tumor cell to producethe truncated Syndecan-2 polypeptide. The truncated Syndecan-2polypeptide can be a dominant negative Syndecan-2 polypeptide (e.g., apolypeptide containing amino acids 1 to 193 of the sequence set forth inSEQ ID NO:2). The tumor cell can be present in a breast tumor, a lungtumor, or a prostate tumor. The method can further include monitoringthe size of the tumor.

The invention also features a method for inhibiting tumor growth. Themethod can include contacting the tumor with a cytoplasmically truncatedSyndecan-2 polypeptide. The contacting can include administering to thetumor a nucleic acid comprising a sequence that encodes thecytoplasmically truncated Syndecan-2 polypeptide. The construct can beexpressed in a cell of the tumor to produce the truncated Syndecan-2polypeptide. The truncated Syndecan-2 polypeptide can be a dominantnegative Syndecan-2 polypeptide (e.g., a polypeptide containing aminoacids 1 to 193 of the sequence set forth in SEQ ID NO:2). The tumor cellcan be present in a breast tumor, a lung tumor, or a prostate tumor. Themethod can further include monitoring the size of the tumor.

In addition, the invention features an antisense polynucleotideeffective to decrease expression from a nucleic acid molecule encoding asyndecan-2 polypeptide. The antisense polynucleotide can be apolynucleotide analogue (e.g., a morpholino-modified polynucleotide).The antisense polynucleotide can contain nucleotide the sequence of SEQID NO:9, SEQ ID NO:10, or SEQ ID NO:11. The syndecan-2 polypeptide canbe a human syndecan-2 polypeptide.

In another aspect, the invention features a cell containing an antisensepolynucleotide effective to decrease expression from a nucleic acidmolecule encoding a syndecan-2 polypeptide.

The invention also features a teleost embryo containing amorpholino-modified antisense polynucleotide effective to decreaseexpression from a nucleic acid molecule encoding a syndecan-2polypeptide, wherein the decreased expression results in an alterationof angiogenesis in the embryo. The teleost embryo can be selected fromthe group consisting of a zebrafish embryo, a stickleback embryo, amedaka embryo, and a puffer fish embryo.

In another aspect, the invention features an isolated nucleic acidhaving the nucleotide sequence of SEQ ID NO:1. The invention alsofeatures an expression vector containing a polynucleotide sequenceoperably linked to an expression control sequence, wherein theexpression control sequence directs production of a transcript from thepolynucleotide sequence, and wherein the transcript is capable ofhybridizing under conditions of high stringency to a target nucleic acidmolecule having the nucleotide sequence of SEQ ID NO:1 or having thecomplement of SEQ ID NO:1.

In yet another aspect, the invention features a purified polypeptidecontaining the amino acid sequence of SEQ ID NO:2.

In still another aspect, the invention features a purified antibody thatbinds specifically to a polypeptide containing the amino acid sequenceof SEQ ID NO:2. The invention also features a method for making anantibody. The method can include immunizing a non-human animal with animmunogenic fragment of a polypeptide having the amino acid sequence ofSEQ ID NO:2. Alternatively, the method can include providing a hybridomacell that produces a monoclonal antibody specific for a polypeptide withthe amino acid sequence of SEQ ID NO:2, and culturing the cell underconditions that permit production of the monoclonal antibody.

In another aspect, the invention features a method for identifying asyndecan-2-modulating agent. The method can include: a) contacting acandidate agent with a living cell preparation producing a syndecan-2polypeptide; b) detecting the amount of syndecan-2 polypeptide in theliving cell preparation subsequent to step (a); and c) identifying thecandidate agent as a syndecan-2-modulating agent if the amount ofsyndecan-2 polypeptide in the living cell preparation is specificallyincreased or decreased relative to a control living cell preparation.

In still another aspect, the invention features a method for identifyingan angiogenesis-modulating agent. The method can include: a) contactingan animal with a syndecan-2-modulating agent; b) monitoring the animalfor any alteration in angiogenesis; and c) identifying thesyndecan-2-modulating agent as an angiogenesis-modulating agent if anyalteration in angiogenesis is detected in step (b).

In another aspect, the invention features a method for making anangiogenesis-modulating agent. The method can include: a) contacting ananimal with a syndecan-2-modulating agent; b) monitoring the animal forany alteration in angiogenesis; c) identifying the syndecan-2-modulatingagent as an angiogenesis-modulating agent if any alteration inangiogenesis is detected in step (b); and d) producing theangiogenesis-modulating agent.

In a further aspect, the invention features a method for promotingangiogenesis in a vertebrate. The method can include administering to avertebrate a functional syndecan-2 polypeptide or a nucleic acidencoding a functional syndecan-2 polypeptide. The vertebrate can be amammal (e.g., a human). The administration can be topical administration(e.g., administration to the skin).

In another aspect, the invention features a method for reducingangiogenesis in a vertebrate. The method can involve administering to avertebrate an antisense polynucleotide effective to decrease expressionfrom a nucleic acid molecule encoding a syndecan-2 polypeptide.

In yet another aspect, the invention features a composition containingan antisense polynucleotide effective to decrease expression from anucleic acid molecule encoding a syndecan-2 polypeptide.

In still another aspect, the invention features a method for detectingsyndecan-2 expression in a tissue. The method can include contacting thetissue with a syndecan-2 probe and detecting binding of the probe to thetissue. The tissue can be a tumor tissue.

The invention also features an antisense polynucleotide effective todecrease expression from a nucleic acid molecule encoding a syndecanpolypeptide. The antisense polynucleotide can be a polynucleotideanalogue (e.g., a morpholino-modified polynucleotide.) The syndecanpolypeptide can be syndecan-2, and the antisense polynucleotide caninclude the nucleotide sequence of SEQ ID NO:9, SEQ ID NO:10, or SEQ IDNO:11.

In another aspect, the invention provides a method for identifying asyndecan-modulating agent. The method can involve (a) contacting acandidate agent with a living cell preparation producing a syndecanpolypeptide, (b) detecting the amount of the syndecan polypeptide in theliving cell preparation subsequent to step (a), and (c) identifying thecandidate agent as a syndecan-modulating agent if the amount of thesyndecan polypeptide in the living cell preparation is specificallyincreased or decreased relative to a control living cell preparation.The syndecan polypeptide can be syndecan-2.

The invention also features a method for identifying anangiogenesis-modulating agent. A method can involve (a) contacting ananimal with a syndecan-modulating agent, (b) monitoring the animal forany alteration in angiogenesis, and (c) identifying thesyndecan-modulating agent as an angiogenesis-modulating agent if anyalteration in angiogenesis is detected in step (b). Thesyndecan-modulating agent can be a syndecan-2-modulating agent.

In another aspect, the invention features a method for making anangiogenesis-modulating agent. The method can involve (a) contacting ananimal with a syndecan-modulating agent, (b) monitoring the animal forany alteration in angiogenesis, (c) identifying the syndecan-modulatingagent as an angiogenesis-modulating agent if any alteration inangiogenesis is detected in step (b), and (d) producing theangiogenesis-modulating agent. The syndecan-modulating agent can be asyndecan-2-modulating agent.

In yet another aspect, the invention features a method for promotingangiogenesis in a vertebrate. The method can involve administering to avertebrate a functional syndecan polypeptide or a nucleic acid encodinga functional syndecan polypeptide. The syndecan polypeptide can besyndecan-2. The vertebrate can be a mammal (e.g., a human). Theadministration can be topical administration (e.g., administration tothe skin).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is the nucleotide sequence of the zebrafish ec2 coding region and5′ untranslated region (SEQ ID NO:1). The start codon is in bold.

FIG. 2 is the amino acid sequence of the zebrafish EC2 polypeptide (SEQID NO:2).

FIG. 3 is an alignment of the mouse (m), rat (r), human (h), Xenopus(X), and zebrafish (z) syndecan-2 polypeptides (SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6, and SEQ ID NO:2, respectively).

FIGS. 4A and 4B are graphs showing the percentage of surviving embryosthat exhibit dorsal curvature following injection with various ec2morpholinos.

FIG. 5 is a graph showing the percentage of surviving embryos thatexhibit dorsal curvature following injection with ec2 and/or nacremorpholinos.

FIG. 6 is a three-dimensional column graph showing the percentage ofsurviving embryos that exhibit a less severe or more severe dorsalcurvature phenotype following injection with ec2 morpholinos.

FIG. 7 is a graph showing the percentage of surviving embryos thatexhibit defects in angiogenesis (“less severe”) or vasculogenesis (“moresevere”) after injection of an ec2 morpholino.

FIG. 8 is a graph showing the percentage of surviving embryos thatexhibit vascular defects after injection with an ec2-MO and/or a VEGFMO.

FIGS. 9A and 9B are graphs showing the percentage of surviving embryosthat exhibit intersegmental expression of flk-1 after injection with anec2-MO in combination with an ec2 expression construct or a controlvector.

FIGS. 10A and 10B are graphs showing the percentage of surviving embryoswith reduced flk-1 expression after injection with vectors encoding acytoplasmically-truncated form of EC2 or full-length EC2, either aloneor in combination with a GFP expression vector or an ec2 morpholino.

FIG. 11 is a graph showing the percentage of surviving embryosexhibiting new sprouts of intersegmental vessels after injection with anec2 morpholino alone or in combination with a human syndecan-2expression construct.

FIG. 12 is a homology tree showing clustering of zebrafish syndecan-2 tothe vertebrate syndecan-2 family.

FIG. 13 is a graph showing the percentages of zebrafish embryos withectopic vessels after injection of a VEGF-165 expression construct aloneor in combination with an expression vector encoding a truncated form ofSyndecan-2 (δS2).

FIG. 14 is a graph showing growth of tumors derived from LCC6 breastcancer cells that were stably transfected with an expression vectorencoding δS2 or vector control (pt2caggs) and inoculated into nude mice.Tumor volumes were measured at weekly intervals.

FIG. 15 is a graph showing tumor vessel density in tumors derived fromLCC6 cells stably transfected with an expression vector encoding δS2 (DNHSYN-2) or vector control (pt2caggs).

FIG. 16A is a graph showing tumor volumes in mice injected with LLC6cells stably transfected with an expression vector encoding δS2. FIG.16B is a graph showing tumor volumes in mice injected with LLC6 cellsstably transfected with a pT2-Luc vector.

DETAILED DESCRIPTION

The discovery that the zebrafish homologue of syndecan-2 (EC2; alsoreferred to herein as Syndecan-2) is involved in angiogenesis indicatesthat angiogenesis can be modulated by increasing or decreasing cellularlevels of functional syndecan polypeptides (e.g., EC2). In addition toec2 nucleic acids and syndecan polypeptides, the subsections belowprovide methods for identifying agents that increase or decrease thebiological effects of syndecans by increasing or decreasing syndecanexpression or by enhancing or inhibiting syndecan function. Similarly,methods for identifying angiogenesis-modulating agents are disclosed;agents that decrease syndecan expression or syndecan function can reduceangiogenesis, for example, and syndecan-2 nucleic acids or syndecanpolypeptides can be used to stimulate angiogenesis. By modulating theexpression or function of syndecans, disease conditions that areassociated with angiogenesis can be managed.

1. Nucleic Acids

As used herein, the term “nucleic acid” refers to both RNA and DNA,including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. A nucleic acid molecule can be double-stranded orsingle-stranded (i.e., a sense or an antisense single strand). Nucleicacids of the invention include, for example, a zebrafish ec2 DNA, whichcan contain the nucleotide sequence of SEQ ID NO:1 and thus encode anEC2 polypeptide having the amino acid sequence of SEQ ID NO:2.

An “isolated nucleic acid” refers to a nucleic acid that is separatedfrom other nucleic acid molecules that are present in a vertebrategenome, including nucleic acids that normally flank one or both sides ofthe nucleic acid in a vertebrate genome (e.g., nucleic acids that flankthe ec2 gene). The term “isolated” as used herein with respect tonucleic acids also includes any non-naturally-occurring nucleic acidsequence, since such non-naturally-occurring sequences are not found innature and do not have immediately contiguous sequences in anaturally-occurring genome.

An isolated nucleic acid can be, for example, a DNA molecule, providedat least one of the nucleic acid sequences normally found immediatelyflanking that DNA molecule in a naturally-occurring genome is removed orabsent. Thus, an isolated nucleic acid includes, without limitation, aDNA molecule that exists as a separate molecule (e.g., a chemicallysynthesized nucleic acid, or a cDNA or genomic DNA fragment produced byPCR or restriction endonuclease treatment) independent of othersequences as well as DNA that is incorporated into a vector, anautonomously replicating plasmid, a virus (e.g., a retrovirus,lentivirus, adenovirus, or herpes virus), or into the genomic DNA of aprokaryote or eukaryote. In addition, an isolated nucleic acid caninclude an engineered nucleic acid such as a DNA molecule that is partof a hybrid or fusion nucleic acid. A nucleic acid existing amonghundreds to millions of other nucleic acids within, for example, cDNAlibraries or genomic libraries, or gel slices containing a genomic DNArestriction digest, is not considered an isolated nucleic acid.

Isolated nucleic acid molecules of the invention can be produced bystandard techniques, including, without limitation, common molecularcloning and chemical nucleic acid synthesis techniques. For example,polymerase chain reaction (PCR) techniques can be used to obtain anisolated ec2 nucleic acid molecule. Isolated nucleic acids of theinvention also can be chemically synthesized, either as a single nucleicacid molecule (e.g., using automated DNA synthesis in the 3′ to 5′direction using phosphoramidite technology) or as a series ofpolynucleotides. For example, one or more pairs of long polynucleotides(e.g., >100 nucleotides) can be synthesized that contain the desiredsequence, with each pair containing a short segment of complementarity(e.g., about 15 nucleotides) such that a duplex is formed when thepolynucleotide pair is annealed. DNA polymerase is used to extend thepolynucleotides, resulting in a single, double-stranded nucleic acidmolecule per polynucleotide pair.

Nucleic acids of the invention can be incorporated into vectors. As usedherein, a “vector” is a replicon, such as a plasmid, phage, or cosmid,into which another nucleic acid segment may be inserted so as to bringabout replication of the inserted segment. Vectors of the inventiontypically are expression vectors containing an inserted nucleic acidsegment that is operably linked to expression control sequences. An“expression vector” is a vector that includes one or more expressioncontrol sequences, and an “expression control sequence” is a DNAsequence that controls and regulates the transcription and/ortranslation of another DNA sequence. Expression control sequencesinclude, for example, promoter sequences, transcriptional enhancerelements, and any other nucleic acid elements required for RNApolymerase binding, initiation, or termination of transcription. Withrespect to expression control sequences, “operably linked” means thatthe expression control sequence and the inserted nucleic acid sequenceof interest are positioned such that the inserted sequence istranscribed (e.g., when the vector is introduced into a host cell).

2. Polynucleotides and Polynucleotide Analogues

“Polynucleotides” are nucleic acid molecules of at least threenucleotide subunits. A nucleotide has three components: an organic base(e.g., adenine, cytosine, guanine, or thymine, herein referred to as A,C, G, and T, respectively), a phosphate group, and a five-carbon sugarthat links the phosphate group and the organic base. In apolynucleotide, the organic bases of the nucleotide subunits determinethe sequence of the polynucleotide and allow for interaction with asecond polynucleotide. The nucleotide subunits of a polynucleotide arelinked by phophodiester bonds such that the five-carbon sugar of onenucleotide forms an ester bond with the phosphate of an adjacentnucleotide, and the resulting sugar-phosphates form the backbone of thepolynucleotide.

“Polynucleotide analogues” are chemically modified polynucleotides. Insome embodiments, polynucleotide analogues can be generated by replacingportions of the sugar-phosphate backbone of a polynucleotide withalternative functional groups. Morpholino-modified polynucleotides,referred to herein as “morpholinos,” are polynucleotide analogues inwhich the bases are linked by a morpholino-phosphorodiamidate backbone(See, Summerton and Weller (1997) Antisense Nuc. Acid Drug Devel.7:187-195; and U.S. Pat. Nos. 5,142,047 and 5,185,444).

In addition to morpholinos, other examples of polynucleotide analoguesinclude analogues in which the bases are linked by a polyvinyl backbone(Pitha et al. (1970) Biochim. Biophys. Acta 204:39-48; Pitha et al.(1970) Biopolymers 9:965-977), peptide nucleic acids (PNAs) in which thebases are linked by amide bonds formed by pseudopeptide2-aminoethyl-glycine groups (Nielsen et al. (1991) Science254:1497-1500), analogues in which the nucleoside subunits are linked bymethylphosphonate groups (Miller et al. (1979) Biochem. 18:5134-5143;Miller et al. (1980) J. Biol. Chem. 255:9659-9665), analogues in whichthe phosphate residues linking nucleoside subunits are replaced byphosphoroamidate groups (Froehler et al. (1988) Nucleic Acids Res.156:4831-4839), and phosphorothioated DNAs, analogues containing sugarmoieties that have 2′ O-methyl groups (Cook (1998) Antisense MedicinalChemistry, Springer, N.Y., pp. 51-101).

Polynucleotides of the invention can be produced through the well-knownand routinely used technique of solid phase synthesis. Equipment forsuch synthesis is commercially available from several vendors including,for example, Applied Biosystems (Foster City, Calif.). Alternatively,other suitable methods for such synthesis can be used (e.g., commonmolecular cloning and chemical nucleic acid synthesis techniques).Similar techniques also can be used to prepare polynucleotide analoguessuch as morpholinos or phosphorothioate derivatives. In addition,polynucleotides and polynucleotide analogues can be obtainedcommercially from, for example, Gene Tools, L.L.C. (Philomath, Org.) orOligos Etc. (Wilsonville, Org.).

Typically, polynucleotide analogues such as morpholinos are singlestranded. Polynucleotide analogues can be of various lengths (e.g., from8 bases in length to more than 112 bases in length, typically from 12 to72 bases in length). Morpholinos can be, for example, 15 to 45 bases inlength (e.g., 18 to 30 bases in length). Polynucleotide analogues can bedesigned to contain certain percentages of each base type (e.g., 40-60%A/T content and 40-60% G/C content, or 50% A/T content and 50% G/Ccontent). In addition, it is particularly useful to avoid sequencescontaining four or more consecutive G residues, as well as secondarystructures such as hairpins.

Polynucleotides and polynucleotide analogues of the present invention(e.g., morpholinos) can be designed to hybridize to a target nucleicacid molecule of known sequence (e.g., a nucleic acid molecule encodingEC2 or another syndecan-2 polypeptide). As described herein, apolynucleotide analogue can have the nucleotide sequence set forth inSEQ ID NO:9, 10, or 11, for example. The term “hybridization,” as usedherein, means hydrogen bonding, which can be Watson-Crick, Hoogsteen, orreversed Hoogsteen hydrogen bonding, between complementary nucleoside ornucleotide bases. For example, A and T, and G and C, respectively, arecomplementary bases that pair through the formation of hydrogen bonds.“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleotides. For example, if a nucleotide at acertain position of a polynucleotide analogue is capable of hydrogenbonding with a nucleotide at the same position of a target nucleic acidmolecule, then the polynucleotide analogue and the target nucleic acidmolecule are considered to be complementary to each other at thatposition. A polynucleotide or polynucleotide analogue and a targetnucleic acid molecule are complementary to each other when a sufficientnumber of corresponding positions in each molecule are occupied bynucleotides that can hydrogen bond with each other. The term“specifically hybridizable” is used to indicate a sufficient degree ofcomplementarity or precise pairing such that stable and specific bindingoccurs between the polynucleotide or polynucleotide analogue and thetarget nucleic acid molecule.

It is understood in the art that the sequence of the polynucleotide orpolynucleotide analogue need not be 100% complementary to that of thetarget nucleic acid molecule to be specifically hybridizable. Apolynucleotide or polynucleotide analogue is specifically hybridizablewhen (a) binding of the polynucleotide or polynucleotide analogue to thetarget nucleic acid molecule interferes with the normal function of thetarget nucleic acid molecule, and (b) there is sufficientcomplementarity to avoid non-specific binding of the polynucleotide orpolynucleotide analogue to non-target sequences under conditions inwhich specific binding is desired, i.e., under conditions in which invitro assays are performed or under physiological conditions for in vivoassays or therapeutic uses.

Hybridization conditions in vitro are dependent on temperature, time,and salt concentration [see, e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory Press, NY (1989)].Typically, conditions of high to moderate stringency are used forspecific hybridization in vitro, such that hybridization occurs betweensubstantially similar nucleic acids, but not between dissimilar nucleicacids. Specific high stringency hybridization conditions arehybridization in 5× SSC (0.75 M sodium chloride/0.075 M sodium citrate)for 1 hour at 40° C. with shaking, followed by washing 10 times in 1×SSC at 40° C. and 5 times in 1× SSC at room temperature.

In vivo hybridization conditions consist of intracellular conditions(e.g., physiological pH and intracellular ionic conditions) that governthe hybridization of polynucleotides and polynucleotide analogues withtarget nucleic acid molecules. In vivo conditions can be mimicked invitro by relatively low stringency conditions. For example,hybridization can be carried out in vitro in 2× SSC (0.3 M sodiumchloride/0.03 M sodium citrate), 0.1% SDS at 37° C. Alternatively, awash solution containing 4× SSC, 0.1% SDS can be used at 37° C., with afinal wash in 1× SSC at 45° C. In order for a polynucleotide orpolynucleotide analogue to specifically decrease expression from atarget nucleic acid molecule, the polynucleotide or polynucleotideanalogue must hybridize specifically to the target nucleic acid moleculeunder physiological conditions.

A polynucleotide or polynucleotide analogue can be complementary to asense or an antisense target nucleic acid molecule. When complementaryto a sense nucleic acid molecule, the polynucleotide analogue is said tobe antisense. When complementary to an antisense nucleic acid molecule,the polynucleotide analogue is said to be sense. For example, apolynucleotide analogue can be antisense to an mRNA molecule or sense tothe DNA molecule from which an mRNA is transcribed. As used herein, theterm “coding region” refers to the portion of a nucleic acid moleculeencoding an RNA molecule that is translated into protein. Apolynucleotide or polynucleotide analogue can be complementary to thecoding region of an mRNA molecule or the region corresponding to thecoding region on the antisense DNA strand. Alternatively, apolynucleotide or polynucleotide analogue can be complementary to thenon-coding region of a nucleic acid molecule. Examples of suchpolynucleotide analogues (morpholinos ec2-MO#2 and ec2-MO#3) aredescribed in Example 4, below. A non-coding region can be, for example,upstream of a transcriptional start site or downstream of atranscriptional end-point in a DNA molecule. A non-coding region alsocan be upstream of the translational start codon or downstream of thestop codon in an mRNA molecule. Furthermore, a polynucleotide orpolynucleotide analogue can be complementary to both coding andnon-coding regions of a target nucleic acid molecule. For example, apolynucleotide analogue can be complementary to a region that includes aportion of the 5′ untranslated region (5′-UTR) leading up to the startcodon, the start codon, and coding sequences immediately following thestart codon of a target nucleic acid molecule. Such a polynucleotideanalogue (morpholino ec2-MO#1) also is described in Example 4, below.

Polynucleotides and polynucleotide analogues of the invention can beuseful for research and diagnostics, and for therapeutic use. Forexample, assays based on hybridization of polynucleotide analogues tonucleic acids encoding EC2 can be used to evaluate levels of EC2 in atissue sample. Hybridization of a polynucleotide analogue of theinvention with a target nucleic acid molecule can be detected by anumber of methods. Some of these methods are well known in the art, andincluding detection by conjugating an enzyme to the polynucleotideanalogues or by radiolabeling of the polynucleotide analogues. Any othersuitable means of detection also can be used. Additionally,polynucleotides and polynucleotide analogues can be employed astherapeutic moieties in the treatment of disease states in animals,including humans (see subsection 6, below).

3. Polypeptides

The invention provides purified syndecan polypeptides. A “polypeptide”refers to a chain of amino acid residues, regardless ofpost-translational modification (e.g., phosphorylation orglycosylation). Proteoglycans therefore also are referred to herein aspolypeptides. Polypeptides of the invention are at least 60 amino acidsin length (e.g., 60, 65, 70, 100, or more than 100 amino acids inlength), and are capable of eliciting a syndecan-specific antibodyresponse (i.e., are able to act as immunogens that induce the productionof antibodies capable of specific binding to a syndecan).

The syndecans make up a class of heparan sulfate proteoglycans. A newlyidentified polypeptide can be classified as belonging to the syndecanfamily of polypeptides based on amino acid sequence comparison withknown syndecan polypeptides. For example, a newly identified polypeptidebelongs to the syndecan class of proteoglycans if it is more similar inamino acid sequence to any member of the syndecan family of polypeptidesthan the two least similar members within the syndecan family. As usedherein, the term “syndecan polypeptide” refers to a polypeptidebelonging to the syndecan class of proteoglycans. The zebrafish EC2polypeptide therefore is a syndecan polypeptide. Furthermore, a syndecanpolypeptide according to the present invention can have the amino acidsequence provided in SEQ ID NO:2, or the amino acid sequence of aportion of SEQ ID NO:2 provided that it is at least 60 amino acids inlength (e.g., at least 60, at least 70, or at least 80 amino acids inlength) and is a syndecan-specific immunogen. As used herein, a“functional syndecan polypeptide” is a syndecan polypeptide that iscapable of promoting angiogenesis (see subsection 6, below).

A syndecan polypeptide can be a dominant negative syndecan polypeptide.For example, a syndecan polypeptide can be a cytoplasmically truncatedform of Syndecan-2. In one embodiment, a cytoplasmically truncated formof Syndecan-2 is encoded by nucleotides 65 to 644 of the sequence setforth in SEQ ID NO:1. This embodiment, designated herein as δS2,contains amino acids 1-193 of the sequence set forth in SEQ ID NO:2, andis a dominant negative form of Syndecan-2. In other embodiments, acytoplasmically truncated form of Syndecan-2 can contain between 180 and205 (e.g., between 185 and 200, or between 190 and 195) amino acids.Such syndecan polypeptides can be useful to inhibit angiogenesis, asdescribed herein.

Syndecan polypeptides can be produced by a number of methods, many ofwhich are well known in the art. By way of example and not limitation,syndecan polypeptides can be obtained by extraction from a naturalsource (e.g., from isolated cells, tissues or bodily fluids), byexpression of a recombinant nucleic acid encoding the polypeptide, or bychemical synthesis.

Syndecan polypeptides of the invention can be produced by, for example,standard recombinant technology, using expression vectors encodingsyndecan polypeptides (e.g., an expression vector containing EC2 codingsequences). Expression vectors can be introduced into host cells (e.g.,by transformation or transfection) for expression of the encodedpolypeptide, which then can be purified. Expression systems that can beused for small or large scale production of syndecan polypeptidesinclude, without limitation, microorganisms such as bacteria (e.g., E.coli and B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA, or cosmid DNA expression vectors containing the nucleicacid molecules of the invention; yeast (e.g., S. cerevisiae) transformedwith recombinant yeast expression vectors containing the nucleic acidmolecules of the invention; insect cell systems infected withrecombinant virus expression vectors (e.g., baculovirus) containing thenucleic acid molecules of the invention; plant cell systems infectedwith recombinant virus expression vectors (e.g., tobacco mosaic virus)or transformed with recombinant plasmid expression vectors (e.g., Tiplasmid) containing the nucleic acid molecules of the invention; ormammalian cell systems (e.g., primary cells or immortalized cell linessuch as COS cells, Chinese hamster ovary cells, HeLa cells, humanembryonic kidney 293 cells, and 3T3 L1 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., the metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter and the cytomegaloviruspromoter), along with the nucleic acids of the invention.

The term “purified” as used herein with reference to a polypeptiderefers to a polypeptide that either has no naturally occurringcounterpart (e.g., a peptidomimetic), or has been chemically synthesizedand is thus uncontaminated by other polypeptides, or has been separatedor purified from other cellular components by which it is naturallyaccompanied (e.g., other cellular proteins, polynucleotides, or cellularcomponents). Typically, the polypeptide is considered “purified” when itis at least 70%, by dry weight, free from the proteins and naturallyoccurring organic molecules with which it naturally associates. Apreparation of the purified polypeptide of the invention therefore canbe, for example, at least 80%, at least 90%, or at least 99%, by dryweight, the polypeptide of the invention.

Suitable methods for purifying the syndecan polypeptides of theinvention include, for example, affinity chromatography,immunoprecipitation, size exclusion chromatography, and ion exchangechromatography. See, for example, Flohe et al. (1970) Biochim. Biophys.Acta. 220:469-476, or Tilgmann et al. (1990) FEBS 264:95-99. The extentof purification can be measured by any appropriate method, including butnot limited to: column chromatography, polyacrylamide gelelectrophoresis, or high-performance liquid chromatography. Syndecanpolypeptides also can be “engineered” to contain a tag sequence (e.g., apolyhistidine tag, a myc tag, or a Flags tag) that allows thepolypeptide to be purified (e.g., captured onto an affinity matrix).Immunoaffinity chromatography also can be used to purify syndecanpolypeptides.

4. Antibodies

The invention also provides antibodies having specific binding activityfor syndecan polypeptides (e.g., EC2 or another syndecan-2 polypeptide).Such antibodies can be useful for detecting levels of the EC2polypeptide in cells treated with morpholinos, for example. Syndecanantibodies also can be useful as syndecan-modulating agents (seesubsection 5, below). As described above, a syndecan polypeptide of theinvention can act as an immunogen to elicit an antibody response that isspecific to EC2, for example, and does not cross-react with a differentpolypeptide. A specific antibody directed to a syndecan polypeptidetherefore will specifically recognize that syndecan, without substantialbinding or hybridizing to other polypeptides that may be present in thesame biological sample.

An “antibody” or “antibodies” includes intact molecules as well asfragments thereof that are capable of binding to an epitope of asyndecan polypeptide. The term “epitope” refers to an antigenicdeterminant on an antigen to which an antibody binds. Epitopes usuallyconsist of chemically active surface groupings of molecules such asamino acids or sugar side chains, and typically have specificthree-dimensional structural characteristics, as well as specific chargecharacteristics. Epitopes generally have at least five contiguous aminoacids. The terms “antibody” and “antibodies” include polyclonalantibodies, monoclonal antibodies, humanized or chimeric antibodies,single chain Fv antibody fragments, Fab fragments, and F(ab)₂ fragments.Polyclonal antibodies are heterogeneous populations of antibodymolecules that are specific for a particular antigen, while monoclonalantibodies are homogeneous populations of antibodies to a particularepitope contained within an antigen. Monoclonal antibodies areparticularly useful.

In general, a syndecan polypeptide is produced as described above, i.e.,recombinantly, by chemical synthesis, or by purification of the nativeprotein, and then used to immunize animals. Various host animalsincluding, for example, rabbits, chickens, mice, guinea pigs, and rats,can be immunized by injection of the protein of interest. Depending onthe host species, adjuvants can be used to increase the immunologicalresponse. These include Freund's adjuvant (complete and/or incomplete),mineral gels such as aluminum hydroxide, surface-active substances suchas lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanin, and dinitrophenol. Polyclonal antibodies arecontained in the sera of the immunized animals. Monoclonal antibodiescan be prepared using standard hybridoma technology. In particular,monoclonal antibodies can be obtained by any technique that provides forthe production of antibody molecules by, for example, continuous celllines in culture as described by Kohler et al. [(1975) Nature256:495-497]; the human B-cell hybridoma technique of Kosbor et al.[(1983) Immunology Today 4:72] and Cote et al. [(1983) Proc. Natl. Acad.Sci. USA 80:2026-2030]; and the EBV-hybridoma technique of Cole et al.[Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. pp. 77-96(1983)]. Such antibodies can be of any immunoglobulin class, includingIgM, IgG, IgE, IgA, IgD, and any subclass thereof. A hybridoma producingthe monoclonal antibodies of the invention can be cultivated in vitro orin vivo.

A chimeric antibody is a molecule in which different portions arederived from different animal species, such as those having a variableregion derived from a mouse monoclonal antibody and a humanimmunoglobulin constant region. Chimeric antibodies can be producedthrough standard techniques.

Antibody fragments that have specific binding affinity for syndecanpolypeptides can be generated by known techniques. Such antibodyfragments include, but are not limited to, F(ab′)₂ fragments that can beproduced by pepsin digestion of an antibody molecule, and Fab fragmentsthat can be generated by deducing the disulfide bridges of F(ab′)₂fragments. Alternatively, Fab expression libraries can be constructed.See, for example, Huse et al. (1989) Science 246:1275-1281. Single chainFv antibody fragments are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge (e.g., 15 to 18amino acids), resulting in a single chain polypeptide. Single chain Fvantibody fragments can be produced through standard techniques, such asthose disclosed in U.S. Pat. No. 4,946,778.

A monoclonal antibody also can be obtained by using commerciallyavailable kits that aid in preparing and screening antibody phagedisplay libraries. An antibody phage display library is a library ofrecombinant combinatorial immunoglobulin molecules. Examples of kitsthat can be used to prepare and screen antibody phage display librariesinclude the Recombinant Phage Antibody System (Pharmacia, Peapack, N.J.)and SurfZAP Phage Display Kit (Stratagene, La Jolla, Calif.).

Once produced, antibodies or fragments thereof can be tested forrecognition of a syndecan polypeptide by standard immunoassay methodsincluding, for example, enzyme-linked immunosorbent assay (ELISA) orradioimmuno assay (RIA). See, Short Protocols in Molecular Biology, eds.Ausubel et al., Green Publishing Associates and John Wiley & Sons(1992). Antibodies that have equal binding affinities for recombinantand native proteins are particularly useful.

5. Syndecan-Modulating Agents

The invention provides methods for identifying substances thatspecifically increase or decrease the amount of a syndecan polypeptidein a cell, tissue, organ, or organism of interest. A substance thatspecifically increases or decreases the amount of a syndecan polypeptideis herein referred to as a “syndecan-modulating agent.” The amount of asyndecan polypeptide in a cell can be assessed by, for example,conventional antibody-based assays. Alternatively, the amount of asyndecan polypeptide can be estimated by detecting syndecan RNA usingconventional nucleic acid-based assays [e.g., northern blotting orreverse transcription-polymerase chain reaction (RT-PCR)]. The amount ofa syndecan polypeptide in a cell can be modulated by increasing ordecreasing the production of syndecan mRNA or the amount of functionalsyndecan polypeptide.

Polynucleotide analogues of the invention can be used to alterexpression from a target syndecan nucleic acid and thus can besyndecan-modulating agents. For example, a morpholino targeted to ec2can be used to decrease production of EC2, while a morpholino targetedto a human syndecan-2 nucleic acid can be used to decrease production ofhuman syndecan-2 protein. As used herein, the term “expression” withrespect to a nucleic acid molecule refers to production of an mRNAmolecule from a DNA molecule as well as production of a polypeptide froman mRNA molecule. Expression from a nucleic acid molecule can bedecreased, for example, by interfering with (1) any process necessaryfor mRNA transcription (e.g., binding of RNA polymerase, binding oftranscription factors, or transcriptional elongation of the mRNA); (2)mRNA processing (e.g., capping or splicing); (3) mRNA transport acrossthe nuclear membrane; or (4) any process necessary for mRNA translation(e.g., ribosome binding or translational initiation, elongation, ortermination). Expression also can be decreased by inducing the cellularnuclease system that degrades cognate mRNAs. In an RNaseH dependentmechanism, for example, a double stranded target mRNA/polynucleotideanalogue is degraded by RNaseH. In addition to polynucleotide analogues,conventional polynucleotides can be used to alter expression from targetnucleic acid molecules to which they are complementary.

As used herein, a “decrease” with respect to expression from a targetnucleic acid molecule refers to a decrease that can be detected byassessing changes in mRNA or protein levels. For example, a decrease canrefer to a 5%, 10%, 25%, 50%, 75%, or more than a 75% decrease inexpression. A decrease in expression also includes complete inhibitionof expression, whereby a 100% decrease in expression from a nucleic acidmolecule is achieved. Changes in mRNA and protein levels can be detectedand/or measured by any of a number of methods known in the art,including but not limited to northern blotting or RT-PCR for mRNAassessment, and western blotting or enzyme-linked immunosorbent assays(ELISA) for protein assessment. Other suitable methods also can be usedto assess mRNA and protein levels.

A decrease in expression from a target syndecan nucleic acid moleculecan be achieved using one polynucleotide analogue. A decrease inexpression from a target syndecan nucleic acid molecule also can beachieved using two polynucleotide analogues having different sequencesand therefore being complementary to different portions of the sametarget nucleic acid molecule. A single polynucleotide analogue can beused to simultaneously decrease expression from two or more syndecannucleic acid molecules that are closely related. In addition, multiplepolynucleotide analogues having sequences complementary to more than onetarget syndecan nucleic acid molecule can be used to decrease expressionfrom multiple target nucleic acid molecules at the same time.

Polynucleotide analogues such as morpholinos can be delivered to aliving cell, tissue, organ, or organism of interest by methods used todeliver single stranded mRNA, such as the methods described in Hyatt andEkker (1999) Meth. Cell Biol. 59:117-126. Non-limiting examples ofdelivery methods include (1) microinjection (e.g., as described inExample 4, below), and (2) simply exposing the cell, tissue, organ, ororganism of interest to the polynucleotide analogue. A cell can be, forexample, a fertilized or unfertilized egg, or a cell in culture. Atissue can be any tissue regardless of its state of differentiation, andcan include, for example, tumor tissue or normal tissue from an organismsuch as a mammal or a fish. An organ can be, for example, thymus, bonemarrow, pancreas, heart, or the blood vessels of the vasculature.Non-limiting examples of organisms include vertebrate embryos such asteleost embryos, juvenile animals, or adult animals. Examples of teleostembryos include zebrafish embryos, puffer fish embryos, medaka embryos,and stickleback embryos.

Polynucleotide analogues can be delivered in a suitable buffer. Asuitable buffer is one in which the polynucleotide analogue can bedissolved, and which is non-toxic to the cell, tissue, organ, ororganism to which the polynucleotide analogue is to be delivered. Anon-toxic buffer can be one that is isotonic to the organism or cell ofinterest. For example, morpholinos can be dissolved in Danieau buffer(see Example 4, below) for injection into zebrafish eggs or embryos.

Alternatively, a polynucleotide designed to hybridize to a targetsyndecan nucleic acid molecule can be inserted into an expression vectorthat is then introduced into the cell, tissue, or organism of interest.For example, a polynucleotide in an expression vector can be operablylinked to an expression control sequence, which will direct theproduction of a polynucleotide transcript that is capable of hybridizingto a target nucleic acid molecule. Methods for introducing a vector intoa cell or an organism are known in the art (e.g., transformation,transfection, and microinjection).

To identify syndecan-modulating agents, a cell that produces syndecanpolypeptides can be contacted with a candidate agent (e.g., a morpholinodesigned to hybridize to a target nucleic acid molecule encoding EC2),and the amount of the syndecan polypeptide or mRNA encoding the syndecanpolypeptide can be determined. A syndecan-modulating agent is one thatcauses an increase or decrease in the amount of syndecan polypeptiderelative to a control cell preparation that was not contacted by thecandidate agent. As described above, the term “increase” or “decrease”refers to any detectable change in the amount of syndecan polypeptide(e.g., a 3%, 6%, 12%, or greater than 12% increase or decrease in theamount of syndecan polypeptide). A syndecan-modulating agent that isspecific will cause an increase or decrease in the functional amount ofonly the polypeptide encoded by the target nucleic acid; polypeptidesencoded by other nucleic acid sequences will not be affected.

Examples of syndecan-modulating agents that decrease levels of syndecanpolypeptides include morpholinos (e.g. those described in the Examples,below) and antibodies against syndecans. Examples of syndecan-modulatingagents that increase levels of syndecan polypeptides include syndecanpolypeptides and nucleic acids encoding syndecan polypeptides.

6. Angiogenesis-Modulating Agents

Angiogenesis refers to the generation of new blood vessels. Under normalphysiological conditions, angiogenesis occurs during wound healing,during tissue and organ regeneration, during embryonic vasculaturedevelopment, and during formation of the corpus luteum, endometrium, andplacenta. Excessive angiogenesis, however, has been associated with anumber of disease conditions. Examples of diseases associated withexcessive angiogenesis include rheumatoid arthritis, atherosclerosis,diabetes mellitus, retinopathies, psoriasis, and retrolentalfibroplasia. In addition, angiogenesis has been identified as a criticalrequirement for solid tumor growth and cancer metastasis. Examples oftumor types associated with angiogensis include rhabdomyosarcomas,retinoblastoma, Ewing's sarcoma, neuroblastoma, osteosarcoma,hemangioma, leukemias, and neoplastic diseases of the bone marrowinvolving excessive proliferation of white blood cells. Due to theassociation between angiogenesis and various disease conditions,substances that have the ability to modulate angiogenesis would bepotentially useful treatments for these disease conditions.

Excessive angiogenesis also can occur during healing at the site of asurgical incision or other tissue trauma, and can result in scarring.Agents with the ability to modulate angiogenesis therefore also would bepotentially useful in treatments to prevent scarring.

The invention provides methods for identifying a substance that (1) is asyndecan-modulating agent, and (2) alters the typical pattern, course,or extent of angiogenesis in a healthy or diseased tissue, organ, ororganism. A syndecan-modulating agent that also alters the typicalpattern, course, or extent of angiogenesis is herein referred to as an“angiogenesis-modulating agent.” An angiogenesis-modulating agent candecrease angiogenesis in a localized tissue or organ, for example in asolid tumor or at the site of a surgical incision. Anangiogenesis-modulating agent also can decrease angiogenesis in asystemic fashion and in some cases, to the extent that no vasculaturedevelopment occurs. For example, a developing zebrafish embryo exposedto an angiogenesis-modulating agent may be devoid of vasculature.Non-limiting examples of angiogenesis-modulating agents that candecrease angiogenesis include polynucleotide analogues directed at ec2nucleic acids and antibodies against EC2. Truncated forms of syndecanpolypeptides also can be used as angiogenesis-modulating agents.

Angiogenesis-modulating agents also can promote or increase angiogenesisin particular situations. For example, it may be desirable to promoteangiogenesis at the site of a surgical incision or other tissue trauma(e.g., at the site of a diabetic skin ulcer). Angiogenesis modulatingagents that promote angiogenesis can be, for example, syndecanpolypeptides or nucleic acid molecules encoding syndecan polypeptides.

The present invention provides pharmaceutical compositions andformulations that include one or more angiogenesis-modulating agents ofthe invention. Pharmaceutical compositions containingangiogenesis-modulating agents can be applied topically (e.g., tosurgical incisions or diabetic skin ulcers). Formulations for topicaladministration of angiogenesis-modulating agents include, for example,sterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions in liquid or solid oilbases. Such solutions also can contain buffers, diluents and othersuitable additives. Formulations for topical administration can includetransdermal patches, ointments, lotions, creams, gels, drops,suppositories, sprays, liquids, and powders. Coated condoms, gloves andthe like also may be useful. Conventional pharmaceutical carriers,aqueous, powder or oily bases, thickeners and the like may be necessaryor desirable. Alternatively, pharmaceutical compositions containingangiogenesis-modulating agents can be administered orally or byinjection (e.g., by subcutaneous, intradermal, intraperitoneal, orintravenous injection).

To identify angiogenesis-modulating agents, an animal can be contactedwith a syndecan-modulating agent and monitored for any alteration orabnormalities in angiogenesis as compared to a control animal that hasnot received the syndecan-modulating agent. Angiogenesis can bemonitored by, for example, microangiography (see Example 7, below). Theanimal can be any vertebrate animal such as a fish, a mouse, a rabbit, aguinea pig, a pig, or a monkey. The animal can be an embryo, a juvenileanimal, or an adult.

The invention also provides methods for using an angiogenesis-modulatingagent to modulate angiogenesis. For example, an angiogenesis-modulatingagent can be administered to a vertebrate (e.g., a zebrafish, a mouse, arat, or a human) such that the level of angiogenesis is altered fromwhat it would be without the angiogenesis-modulating agent. In someembodiments, the angiogenesis-modulating agent is administered in anamount effective to reduce angiogenesis. As used herein, a “reduction”in the level of angiogenesis in a vertebrate treated with anangiogenesis-modulating agent refers to any decrease (e.g., a 1%decrease, a 5% decrease, a 10% decrease, a 25% decrease, a 50% decrease,a 75% decrease, a 90% decrease, or a 100% decrease) in the level ofangiogenesis in the treated vertebrate as compared to the level ofangiogenesis in an untreated vertebrate. For example, an antisensepolynucleotide analog such as a morpholino directed against syndecan-2can be administered to a vertebrate in order to reduce the level ofangiogenesis (see, e.g., Example 7, below). Alternatively, more than oneangiogenesis-modulating agents can be administered to a vertebrate toreduce angiogenesis. For example, a morpholino targeted to syndecan-2and a morpholino targeted to another nucleic acid (e.g., a nucleic acidencoding vascular endothelial growth factor, or VEGF) can beadministered simultaneously or sequentially to a vertebrate to reducethe level angiogenesis in a vertebrate (see, e.g., Example 7).

A truncated form of a syndecan (e.g., a cytoplasmically truncated formof a syndecan, such as the δS2 form of Syndecan-2) also can beadministered to a vertebrate to reduce the level of angiogenesis. Insome embodiments, one or more angiogenesis-modulating agents can beadministered directly to a tumor or a tumor cell in a vertebrate (e.g.,a breast tumor, a lung tumor, or a prostate tumor). Such administrationcan result in decreased angiogenesis in the tumor, and can kill thetumor cell or prevent or reduce growth of the tumor. The truncated formof syndecan can be administered in polypeptide form. Alternatively, avertebrate or a tumor can be contacted with a nucleic acid containing asequence that encodes the truncated form of the syndecan, such that thecoding sequence is expressed to produce the truncated syndecan. Methodsof the invention also can include monitoring the size of the tumor,before and/or after administration of the truncated syndecan.

In other embodiments, the angiogenesis-modulating agent can increaseangiogenesis. As used herein, an “increase” in the level of angiogenesisin a vertebrate treated with an angiogenesis-modulating agent refers toany increase (e.g., a 1% increase, a 5% increase, a 10% increase, a 25%increase, a 50% increase, a 75% increase, a 90% increase, a 100%increase, or more than a 100% increase) in the level of angiogenesis inthe treated vertebrate as compared to the level of angiogenesis in anuntreated vertebrate. For example, a functional syndecan-2 polypeptideor a nucleic acid encoding a functional syndecan-2 polypeptide can beadministered to a vertebrate to increase angiogenesis.

7. Diagnostic and Prognostic Applications

The invention also provides methods for using syndecan probes to detectsyndecan expression in a cell preparation or in a particular tissue. Forexample, a technique such as in situ hybridization with a syndecan-2nucleic acid probe can be used to detect syndecan-2 mRNA in a tissue(e.g., a tumor tissue; see Example 13, below). Such probes can belabeled with a variety of markers, including radioactive,chemiluminescent, or fluorescent markers, for example. Alternatively, animmunohistochemistry technique with an anti-syndecan-2 antibody can beused to detect syndecan-2 protein in a cell or a tissue. As syndecan-2has been implicated in angiogenesis and vasculogenesis, the level ofsyndecan-2 mRNA or protein expression could serve as a diagnostic orprognostic indicator of cancer. For example, a tumor tissue exhibiting ahigher level of syndecan-2 expression may have a more developedvasculature, and thus may be more likely to metastasize than a tumortissue with less syndecan-2 expression.

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1 Identification of a Zebrafish Gene, ec2, Encoding aSyndecan-2 Homologue

Using BLAST analysis, a clone containing a coding sequence with strongsimilarity to mouse and human syndecan-2 was identified in a zebrafishEST database (GenBank accession number AI558535). The coding sequence,corresponding to a zebrafish syndecan-2 gene, was named ec2.

To obtain the full-length zebrafish ec2 coding sequence, automaticsequencing reactions were performed using primers based on the partialsequence reported in the EST database. The primers used to obtain thecomplete ec2 cDNA sequence were 5′-GAAGATCTCACCATGAGGAACCTTTGGATGAT-3′(SEQ ID NO:7), and 5′-GAAGATCTTTATGCGTAAAACTCCTTGG-3′ (SEQ ID NO: 8).

The full-length sequence of the zebrafish ec2 open reading frame,together with the 5′-UTR, is shown in FIG. 1 (SEQ ID NO: 1). Thepolypeptide sequence of zebrafish EC2 (SEQ ID NO:2; FIG. 2) has 48% to50% sequence identity with human and mouse syndecan-2, as determined byclustal alignment using the GeneWorks v. 2.5.1 software. The alignmentof zebrafish EC2 with mouse, rat, human, and Xenopus syndecan-2 is shownin FIG. 3.

Example 2 Zebrafish Care and Egg Collection

Standard zebrafish care protocols are described in Westerfield (2000)The zebrafish book: A guide for the laboratory use of zebrafish (Daniorerio), 4^(th) ed., University of Oregon Press, Eugene.

Zebrafish were kept in 6.5 gallon (26 liter) and 20 gallon (76 liter)plastic tanks at 28° C. Tanks with a 6.5 gallon capacity housed 25 fish,while 20 gallon tanks housed 70 fish. Tank water was constantly changedwith carbon-filtered, UV-sterilized tap water (system water) at a rateof 15 to 40 mL/min, or was replaced each day by siphoning debris fromthe bottom of the tank. Tap water that had aged at least one day in anopen (heated) tank to release chlorine was adequate, although moreconsistent conditions were obtained by adding commercial sea salts todeionized or distilled water (60 mg of Instant Ocean® salt per liter ofwater; see Westerfield, supra). A 10-hour dark and 14-hour light daycycle was maintained in the zebrafish facility.

Fish were fed brine shrimp twice a day. To make shrimp, 100 mL of brineshrimp eggs were added to 18 L of salt water (400 mL of Instant Ocean®salt per 18 L of water) and aerated vigorously. After 2 days at 28° C.,the shrimp were filtered through a fine net, washed with system water,suspended in system water, and fed to fish. Alternatively, fish couldalso be fed with ‘Tetra’ brand dry flake food.

Zebrafish spawning was induced every morning shortly after the start ofthe light cycle. To collect the eggs, a ‘false bottom container’ systemwas used (Westerfield, supra). The system consisted of two containers ofapproximately 1.5 L, one slightly smaller than the other. The bottom ofthe smaller container was replaced with a stainless steel mesh havingholes bigger than the diameter of zebrafish eggs. The smaller containerwas placed into the bigger container, and the setup was filled withsystem water. Up to eight zebrafish were placed inside the smallercontainer. When the fish spawned, the eggs fell through the mesh intothe bigger container and thus could not be reached by the fish andeaten. About 10-15 minutes were allowed for spawning, after which timethe smaller container with the fish was transferred into a second biggercontainer. Eggs were collected by filtering the remaining contents ofthe first bigger container through a mesh having holes smaller than thediameter of the eggs. Fish were used for spawning once a week foroptimal embryo production.

Example 3 Spatial Expression Pattern of ec2 in Early Zebra Fish Embryos

In situ hybridization was performed to determine the expression patternof ec2 during zebrafish embryo development. The zebrafish ec2 codingregion and 5′-UTR was labeled with digoxigenin-UTP (Roche Diagnostics,Indianapolis, Ind.) and used as a probe. In situ hybridization wasperformed as described in Jowett et al. (Jowett et al. (1999) MethodsCell Biol. 59:63-85). The spatial expression pattern of ec2 wasdetermined during late somitogenesis (20.5 hours post-fertilization), atthe 26-somite stage (22 hours post-fertilization), at the 28-somitestage (23 hours post-fertilization), and at time pointspost-somitogenesis (27, 28, 33, and 48 hours post-fertilization). At20.5 hours, ec2 expression was observed in the vascular mesenchyme, orcells surrounding the presumptive axial vessels. At the 26-somite stage,ec2 expression also was detected in the hypochord, a single cell-widemidline structure immediately ventral to the notochord and dorsal to thedorsal aorta. This expression pattern persisted through 33 hourspost-fertilization but had disappeared by 48 hours, at which point ec2expression was detected in the dorsal fin buds. Expression of ec2 alsowas detected throughout the head and the dorsal neural tube starting atabout 22 hours post-fertilization, suggesting a possible function of EC2in development of the central nervous system.

Example 4 Morpholino Inactivation of Zebrafish ec2

To determine the function of ec2 in early zebrafish development,morpholinos (MOs) targeting the 5′-UTR of zebrafish ec2 were generatedand used to decrease EC2 production. The zebrafish ec2-MOs had thefollowing sequences: ec2-MO#1: 5′-GGTTCCTCATAATTCCTCAGTCTTC-3′ (SEQ IDNO:9) ec2-MO#2: 5′-GCTCGTGAAAGCGGAAAATCGC-3′ (SEQ ID NO:10) ec2-MO#3:5′-CCTCAGTCTTCGCTCGTGAAAGCG-3′ (SEQ ID NO:11)

In addition, an ec2-MO with a 4-base mismatch to ec2-MO#1, designatedec2-MO (Δ4), was used to assess the specificity of ec2-MO targeting.ec2-MO (Δ4) had the following sequence: 5′-GGTaCCTgATAATaCCTCAcTCTTC-3′(SEQ ID NO:12). The mismatched bases are indicated by lowercase letters.As other negative controls, a nacre-MO (5′-CATGTTCAACTATGTGTTAGCTTCA-3′,SEQ ID NO:13) and a UROD-MO (5′-GAATGAAACTGTCCTTATCCATCA-3′, SEQ IDNO:14) were generated.

Morpholinos were obtained from Gene Tools, L.L.C. (Philomath, Org.), andwere designed to bind to the 5′-UTR at or near the initiatingmethionine. Sequences were selected based on parameters recommended bythe manufacturer, such that morpholinos were 21 to 25 nucleotides inlength and had 50% G/C and 50% A/T content. Internal hairpins and runsof four consecutive G nucleotides were avoided.

Morpholinos were solubilized in water at a concentration of 50 mg/mL.The resulting stock solution was diluted to working concentrations of0.09 to 3 mg/mL in water or 1× Danieau solution. Danieau bufferconsisted of 8 mM NaCl, 0.7 mM KCl, 0.4 mM MgSO₄, 0.6 mM Ca(NO₃)₂, and5.0 mM HEPES (pH 7.6). Zebrafish embryos at the 1 to 4 cell stages weremicroinjected with 4-9 nL of morpholinos.

The morpholino injection method was very similar to the mRNA injectionmethod described in Hyatt and Ekker (supra). The collected eggs weretransferred onto agarose plates as described in Westerfield (supra).While agarose plates for mRNA injections were kept cold to slow embryodevelopment, the plates for morpholino injections were prewarmed toapproximately 20° C., since morpholino injection into cold embryos wasfound to increase non-specific effects and mortality of the injectedembryos.

Needles used for morpholino injections were the same as for mRNAinjections (Hyatt and Ekker, supra). The needles were back-filled with apipette and calibrated by injecting the loaded morpholino solution intoa glass capillary tube. The picoinjector volume control was then set upfor 1.5 to 15 nL. The injection volume depended on the required dose;1.5 ng to 18 ng of morpholino usually were injected. Morpholinosolutions were injected through the chorion into the yolk of zebrafishembryos. Injected embryos were transferred to petri dishes containingsystem water and allowed to develop at 28° C. Typically, at least 80% ofthe embryos injected in each experiment survived and were used forsubsequent experiments.

Example 5 Efficacy of Morpholino Targeting

To assess the efficacy of morpholino targeting of ec2, an ec2 5′untranslated region-green fluorescent protein (UTR-GFP) fusion constructwas prepared with the ec2 5′ UTR containing the ec2-MO#1 targetingsequence. PCR mutagenesis was used to amplify 5′ ec2 sequences (primers5′-GCAGGATCCGCGATTTTCCGCTTTCACGA-3′, SEQ ID NO:15; and5′-ACCTGAATTCAGGTTCCTCATAATTCCTCAG-3′, SEQ ID NO:16) and GFP sequences(primers 5′-ACGTGAATTCGAGTAAAGGAGAAGAACTT-3′, SEQ ID NO:17; and5′-CAGTCTCGAGTTATTTGTATAGTTCATCCATG-3′, SEQ ID NO:18). The ec2 and GFPamplicons were digested with EcORI/XhoI and BamHI/EcORI, respectively,and subcloned into pCS2+(Rupp et al. (1994) Genes Dev. 8:1311-1323; andTurner and Weintraub (1994) Genes Dev. 8:1434-1447) to generate thepCS2+ec2 5′ UTR-GFP fusion construct.

The fusion construct was linearized with NotI. SP6 RNA polymerase(Ambion, Austin, Tex.) was used for in vitro synthesis of mRNA. Embryoswere co-injected with mRNA synthesized from the fusion construct andec2-MO#1 or the UROD MO as a negative control. GFP expression wasassessed as previously described (Nasevicius and Ekker (2000) Nat.Genet. 26:216-220). Injection of embryos with both 7 ng ec2-MO#1 and theec2 5′ UTR-GFP RNA resulted in a drastic reduction in GFP expression ascompared to the level of GFP expression in embryos injected only withthe ec2 5′ UTR-GFP RNA. In contrast, co-injection with the UROD MOresulted in strong GFP expression at a level comparable to that observedin embryos injected only with the ec2 5′ UTR-GFP RNA.

Example 6 Morphology of Zebrafish Embryos Injected with ec2-MO

The phenotypes of zebrafish embryos injected with morpholinos were firstassessed by visual inspection with a dissecting microscope. At about 24hours post-fertilization, embryos began to exhibit varying extents ofdorsal curvature. Approximately 7-8% of the embryos that survived afterinjection of 5 ng ec2-MO#1 or 6 nm ec2-MO#2 exhibited dorsal curvatureat 24 hours post-fertilization (FIG. 4A). When ec2-MO#1 and ec2-MO#2were injected together, slightly more than 40% of the surviving embryoshad dorsal curvature. In separate studies, 50% of embryos injected with8 ng ec2-MO#1 displayed dorsal curvature at 24 hours (n=63), as did 12%of embryos injected with 6 ng ec2-MO#2 (n=85) and 35% of embryosinjected with 7.5 ng ec2-MO#3 (n=69; FIG. 4B). Injection with 8 ng ofthe 4-base mismatch morpholino, ec2-MO (Δ4), did not result in a curvedphenotype in any 24 hour embryos. In addition to dorsal curvature,embryos injected with the ec2 morpholinos exhibited an enlargedpericardium, a lack of visible circulation, and defective headformation, possibly due to cell death in the brain.

The effect of the ec2 morpholinos was specific, as injection of eitherec2-MO#1, ec2-MO#2, or ec2-MO#3 gave rise to the same phenotype whileec2-MO (Δ4) had no effect. Furthermore, injection with 7 ng of thenacre-MO did not result in any embryos with dorsal curvature (FIG. 5),and the nacre-MO did not synergize with ec2-MO#1 to increase theincidence of the curved phenotype above the level observed with ec2-MO#1alone.

The appearance of the dorsal curvature in affected embryos ranged from amild bend to a more extreme, U-shaped curve. Embryos exhibiting dorsalcurvature thus were scored as having a less severe or a more severephenotype. As depicted in FIG. 6, most surviving embryos with dorsalcurvature after injection of either ec2-MO#1 or ec2-MO#2 displayed theless severe phenotype. Simultaneous injection of both ec2-MO#1 andec2-MO#2 caused approximately 30% of the injected embryos to display theless severe phenotype and slightly more than 5% of the embryos todisplay the more severe phenotype. These morpholinos therefore actsynergistically.

Example 7 Microangiography Analysis of Zebrafish Embryos Injected withec2-MOs

To determine whether the vasculature in zebrafish embryos injected withec2-MOs formed properly, microangiography was performed on bothuninjected control embryos and embryos injected with ec2-MOs.Fluorescein isothiocyanate-(FITC-) Dextran dye was microinjected intothe common cardinal vein of zebrafish embryos as described in Naseviciuset al. (2000) Yeast 17:294-301. Between 10-15 nL of FITC-Dextranfluorescent dye (1 μg/mL) was microinjected into 48 hour embryosincubating in 0.004% Tricain solution. The dye was taken to the heartand then pumped into the systemic circulation, allowing visualization ofthe entire vasculature by fluorescent microscopy. These studies revealedthat nearly 100% of embryos injected with 8 ng ec2-MO#1 exhibiteddefects in angiogenesis (sprouting of new vessels from existing axialvessels) and/or vasculogenesis (initial formation of axial vessels).FIG. 7 shows the percentage of surviving injected embryos that exhibiteda less severe phenotype vs. a more severe phenotype at 48 hourspost-fertilization. Embryos with defective angiogenesis were scored ashaving a less severe phenotype, while those with defectivevasculogenesis were scored as having a more severe phenotype. Between 25and 31 surviving injected embryos were scored in each experiment. In theleast severe cases, intersegmental vessels failed to form and thevascular plexus in the tail region failed to develop into a more complexnetwork as seen or uninjected wild type embryos. In the most severecases, no circulation was observed.

To further assess the nature of vascular defects in ec2-MO injectedembryos, histological analysis was performed on those embryos showing nocirculation upon microangiography analysis. Transverse sections wereobtained from uninjected wild type embryos and from embryos injectedwith 8 ng ec2-MO#1 that showed no circulation. The sections were stainedwith hematoxylin and eosin. Compared to wild type embryos, the ec2-MOinjected embryos exhibited a severely dilated dorsal aorta, suggestingthat functional blood vessels had failed to form.

In other experiments, MOs targeted to ec2 and VEGF were injectedsimultaneously. The VEGF-A MO#1 had the sequence5′-GTATCAAATAAACAACCAAGTTCAT-3′ (SEQ ID NO:19). Embryos were injectedwith 1 ng of ec2-MO#1 or 0.5 ng of VEGF-A MO#1 alone, or co-injectedwith 1 ng of ec2-MO#1 and 0.5 ng of VEGF-A MO#1 or 1 ng of ec2-MO (A4)and 0.5 ng of VEGF-A MO#1. Injected embryos were analyzed for vasculardefects at 48 hours post-fertilization by microangiography. As shown inFIG. 8, a low dose of ec2-MO#1 and a low dose of VEGF-A MO#1 interactedsynergistically in causing angiogenic defects in co-injected embryos.Microangiography revealed a weak defect in sprouting of intersegmentalvessels in embryos injected with ec2-MO#1 alone, and a weak sproutingdefect in the anterior trunk of embryos injected with VEGF-A MO#1 alone.Co-injected embryos exhibited more severe defects, such as aberrantsprouting of intersegmental vessels or even no sprouting of vessels inthe trunk. No significant interaction between ec2-MO (Δ4) and VEGF-AMO#1 was observed.

Example 8 Expression of Early and Late Vascular Markers After Injectionwith ec2-MOs

In situ hybridization experiments were performed to assess theexpression of vascular markers in embryos injected with ec2-MOs and inuninjected controls. Expression of the early vascular markers, fli-1 andflk-1, was examined at 24 hours post-fertilization, while expression ofthe late vascular markers, tie-1 and tie-2, was examined between 25 and48 hours post-fertilization. Axial expression of both flk-1 and fli-1was retained at 24 hours in both control and ec2-MO injected embryos,but intersegmental expression was absent in 76% of embryos injected with8 ng ec2-MO#1 (n=24). This suggests that the process of angiogenicsprouting did not occur in the ec2-MO injected embryos. Morespecifically, 82% of the embryos injected with 8 ng ec2-MO#1 had reducedlevels of fli-1 as compared to controls, while 75%±0% displayed reducedlevels of flk-1.

To assess the integrity of vasculogenesis, ephrin-B2 and ephrin-B4expression was analyzed in embryos injected with 8 ng ec2-MO#2.Ephrin-B2 and Ephrin-B4 are transmembrane ligands that mark arterial andvenous endothelial cells, respectively (Wang et al. (1998) Cell93:741-753). Expression of ephrin-B2 in the dorsal aorta was notaffected in ec2-MO injected embryos. Expression of ephrin-B4 also wasnormal, suggesting that primary formation of the axial vessels wasnormal in ec2-MO injected embryos.

Intersegmental expression of tie-1 was reduced at about 25-26 hours in68% of embryos injected with 8 ng ec2-MO#1 (n=27), as compared touninjected controls. A reduction in axial expression was observed asearly as 28 hours post-fertilization. 43%+3% of injected embryos showedlower levels of the tie-2 at 48 hours, as compared to uninjectedembryos. Expression of tie-2 also was reduced at 28 hours in 62% ofembryos injected with 8 ng ec2-MO#1, and remained reduced at 48 hours.Thus, EC2 may play an important role in stabilization and maintenance ofmature vessels.

Example 9 Rescue of MO-Induced Angiogenic Defects by Exozenous EC2Protein

Experiments were conducted to determine whether the presence ofexogenous zebrafish EC2 protein could rescue the angiogenic defectobserved in ec2-MO injected embryos. An EC2 expression construct wasprepared by using PCR to introduce EcORI sites into the 5′ and 3′ endsof the zebrafish ec2 open reading frame. A plasmid containing the ec2coding sequence was used as a template. The primers (with EcORI sites attheir 5′ ends) were: 5′-CCGGAATTCCACCATGAGGAACCTTTGGATGAT-3′, SEQ IDNO:20; and 5′-CCGGAATTCTTATGCGTAAAACTCCTTG-3′, SEQ ID NO:21. The PCRfragment was subcloned into the EcORI site of the FRM 2.1 expressionvector (Gibbs et al. (2000) Marine Biotechnology 2:107-125).

Embryos were injected with 7-8 ng of ec2-MO#3 and 3 pg of the EC2expression construct, and a subset also was injected with a solution ofthe ec2 5′ UTR-GFP fusion construct. GFP expression was used as alineage tracer to facilitate the identification of successfully injectedembryos. In situ analysis of flk-1 expression revealed that asignificantly higher fraction of embryos co-injected with ec2-MO and ec2DNA showed intersegmental vessels (FIG. 9A). In contrast, there was nosignificant difference in the fraction of embryos showing intersegmentalexpression of flk-1 in embryos co-injected with ec2-MO and the GFPexpression construct compared to those injected with ec2-MO alone (FIG.9B). These experiments indicated that the angiogenic defect observed inec2-MO-injected embryos is specific to a loss of function of theendogenous ec2 gene.

Example 10 Forced Expression of Cytoplasmically-Truncated EC2

To determine whether a cytoplasmically-truncated form of EC2, δS2, wouldhave a deleterious effect on vascular development in zebrafish embryos,δS2 was overexpressed in embryos by injection of a δS2 expressionconstruct. To generate this expression construct, a DNA fragmentencoding a cytoplasmically truncated form of zebrafish EC2 was generatedby PCR using plasmid DNA containing the ec2 coding sequence as atemplate. The primers (with EcORI sites at their 5′ ends) were5′-CCGGAATTCCACCATGAGGAACCTTTGGATGAT-3′ (SEQ ID NO:22, and5′-CCGGAATTCTTACGGTTTCCTCTCTCCCAG-3′ (SEQ ID NO:23). The PCR fragmentwas subcloned into the EcORI site of the FRM 2.1 expression vector.

Embryos were injected with either 9 pg GFP expression construct alone ora mixed solution of 8 pg δS2 and 1 pg GFP expression construct, andassessed for possible vascular defects by microangiography and molecularanalyses. Forced expression of δS2 at lower doses did not affectmorphology, but defective angiogenic sprouting in the trunk was observedupon microangiography analysis. In situ analysis of flk-1 expressionindicated reduced sprouting in δS2-injected embryos, mimicking theeffect of ec2-MO injections (FIG. 10A). In contrast, forced expressionof EC2 and GFP did not have any significant effect on angiogenicsprouting. In other experiments, embryos were injected with 1 ngec2-MO#3, 1.5 pg δS2 expression construct, 1.5 pg δS2 expressionconstruct plus 1 ng ec2-MO#3, or 1.5 pg EC2 expression construct plus 1ng ec2-MO#3. These studies revealed that a low dose of δS2 enhanced theeffect of injecting a low dose of ec2-MO (FIG. 10B). Thus, forcedexpression of δS2 in embryos mimics the angiogenic defect observed inec2-MO injected embryos, and support the anti-morphic function of δS2 inangiogenesis.

Example 11 The Vascular Function of Syndecan-2 is Conserved

Both zebrafish and mouse syndecan-2 are embryonically expressed inmesenchymal cells surrounding the axial vessels, suggesting that thevascular function of syndecan-2 is conserved. The functionalconservation of syndecan-2 was tested in vascular development byassessing whether human syndecan-2 proteins could rescue the angiogenicdefect in ec2-MO injected embryos. To prepare a human syndecan-2expression construct, the open reading frame was amplified from a humanfetal liver cDNA library (Genemed Biotechnologies, Inc.) using thefollowing primers: 5′-ATGCGGCGCGCGTGGATC-3′ (SEQ ID NO:24), and5′-TTACGCATAAAACTCCTTAGTAG-3′ (SEQ ID NO:25). The primers were designedbased on the human syndecan sequence found in GenBank Accession No.XM_(—)040582. EcORI sites were introduced at the 5′ and 3′ ends of thecoding sequence by another round of PCR. The PCR fragment wassubsequently subcloned into the EcORI site of the FRM expressionconstruct.

Embryos were injected with 7-8 ng of ec2-MO#3 alone or in combinationwith the 4-5 pg of the human syndecan-2 expression construct.Intersegmental expression of flk-1 was analyzed in situ at 24 hourspost-fertilization to assess the degree of angiogenic sprouting in thetrunk. A significantly higher percentage of the group co-injected withthe ec2-MO and human syndecan-2 DNA exhibited new sprouts, as comparedto the group injected with ec2-MO alone (FIG. 11). In addition, asignificantly higher fraction of embryos that were co-injected withec2-MO and the human syndecan-2 expression construct showedintersegmental expression of flk-1, compared to those injected withec2-MO only. The observation that human syndecan-2 protein alleviatedthe angiogenic defect observed in ec2-MO injected embryos suggests thatthe vascular function of syndecan-2 is conserved.

Example 12 Syndecan-2 Function in Vertebrates

Based on amino acid sequence comparison, zebrafish EC2 uniquely clustersto the vertebrate syndecan-2 family (FIG. 12). To address whethersyndecan-2 might perform similar vascular functions in other vertebrateorganisms, expression of syndecan-2 was analyzed in mouse embryos atstages of development similar to those analyzed in zebrafish asdescribed herein. On embryonic day 9.5, mouse syndecan-2 was expressedstrongly in the head region and in the mesenchyme around axial vessels,similar to the expression pattern of ec2 in zebrafish embryos at 24hours post-fertilization. This conservation of syndecan-2 expression inmouse suggests that syndecan-2 performs an essential vascular functionduring mammalian embryonic development.

Example 13 Expression of Human Syndecan-2 in Tumor Tissues

A survey of syndecan-2 expression was in various tumor tissues wasperformed using tissue spotted onto multi-tumor tissue microarray slidesobtained from the Cooperative Human Tissue Network at National CancerInstitute (Bethesda, Md.). Tumor samples from eight tumor types (braintumor, breast adenocarcinoma, colonic adenocarcinoma, lung cancer,lymphoma, melanoma, ovarian adenocarcinoma and prostate adenocarcinoma)were spotted on each slide. In situ hybridization was performed on theslides, using DIG-labelled human syndecan-2 RNA as a probe. A 390 bpfragment containing the partial human syndecan-2 coding sequence wasamplified using the human syndecan-2 expression construct as thetemplate. The primers used were 5′-ATGCGGCGCGCGTGGATC-3′ (SEQ ID NO:26),and 5′-CATTTGTACCTCTTCGGCTG-3′ (SEQ ID NO:27). The PCR fragment wassubcloned into the TOPO vector (Invitrogen, Carlsbad, Calif.). Theplasmid DNA was linearized with NotI, and T3 RNA polymerase was used forin vitro synthesis of a DIG-labelled anti-sense probe. Tumor slides weredehydrated through a 100-90-70-30 ethanol series, 10 minutes each. Insitu hybridization was performed using a protocol provided by the Chuanglab website (“baygenomics” dot “ucsf” dot “edu” slash “protocols”).

Syndecan-2 expression was detected in 15 samples representing breastadenocarcinoma, lung squamous carcinoma and prostate adenocarcinomatumor types. Positive staining was observed in and around tumor bloodvessels in some of those samples. Expression of syndecan-2 in selectivetumor tissue vasculature strongly suggests its potential function intumorigenesis as an angiogenic agent.

Example 14 Effects of δS2 on VEGF-Induced Angiogenesis

An FRM expression vector encoding δS2 was generated using the followingprimers: 5′-ATGAGGAACCTTTGG ATGAT-3′ (SEQ ID NO:28) and5′-TTACGGTTTCCTCTCTCCCTA-3′ (SEQ ID NO:29). A DNA fragment containingthe VEGF-165 open reading frame was isolated from the CS2+-VEGF-165plasmid (Ruowen Ge) by EcORI restriction digest. The purified DNAfragment was subcloned into the EcORI site of the FRM expressionconstruct. The test DNA constructs (5 pg of the VEGF-165 constructalone, or 5 pg of the VEGF-165 construct with 2 pg of the δS2 construct)were mixed with an FRM-enhanced GFP (EGFP) expression construct. Themixtures were injected into 1-cell embryos at the interface between theyolk and the blastomere to ensure uniform mosaic distribution. Embryosshowing GFP expression were selected prior to fixation for subsequent insitu analysis.

Microangiography analysis as described in Example 7 revealed that nearly50% of the embryos injected with the VEGF-165 expression construct alonehad ectopic vessels (FIG. 13). Co-injection with the δS2 expressionconstruct significantly decreased the occurrence of ectopic vessels.Thus, the dominant negative Syndecan-2 was able to inhibit VEGF-inducedactivation of angiogenesis.

Example 15 Effects of δS2 on Tumors Derived from LCC6 Breast CancerCells

Cell culture: Breast cancer-derived LCC6 cells (Doug Yee, University ofMinnesota) were cultured in high glucose DMEM (Gibco/Invitrogen,Carlsbad, Calif.) supplemented with 10% FBS and 1%penicillin/streptomycin (Gibco/Invitrogen).

Cloning of human syndecan-2 expression constructs: The DNA fragmentcontaining the human syndecan-2 coding sequence was isolated from theFRM-syndecan-2 expression construct (Chen et al. (2004) Blood103:1710-1719) by EcORI digestion. The cytoplasmically truncated form ofSyndecan-2 (δS2; amino acids 1-186) was amplified by PCR using thepT2caggs-syndecan-2 expression construct as the template and primershaving the sequences set forth in SEQ ID NO:22 and SEQ ID NO:23, with apremature stop codon introduced at the 3′ end. Each DNA fragment wassubcloned into the EcORI site of the pT2caggs plasmid (DavidLargaespada, University of Minnesota).

Transfection: LCC6 cells were co-transfected with the test plasmid(pT2caggs empty vector or pT2caggs-human syndecan-2 derivatives) and amarker plasmid (pT2-CMV-GFP), using the ExGen 500 (linearpolyethylenimine based) transfection reagent (Fermentas). Cells werechanged to selection media (200 ng/mL puromycin) two days aftertransfection. About 20 days later, individual stably transfected cloneswere selected for expansion. Expression of the transgene in stableclones was assessed using RT-PCR.

RT-PCR analysis: Total RNA was isolated from 1×10⁶ cells. To assessexpression of endogenous syndecan-2 in each cell line, the followingprimers were used: 5′-ACCTTGACAACAGCTCCATT-3′ (SEQ ID NO:28) and5′-AGACTGTCTGAGTGT TTCTC-3′ (SEQ ID NO:29). To assess expression oftransgenic syndecan-2 derived mRNA, the following transgene-specificprimers were used: 5′-TGAGAAACACTCAG ACAGTCT-3′ (SEQ ID NO:30) and5′-CTCAAGGGGCTTCATGATG-3′ (SEQ ID NO:31). The reverse primer anneals toa 3′ untranslated region downstream of the syndecan-2 derived transgenein the pT2caggs plasmid. Human GAPDH expression was used as loadingcontrol. Primers used were: 5′-CCACCCATGGCAAATTCCATGGCA-3′ (SEQ IDNO:32) and 5′-TCTAGACGGCAGGTCAGGTCCACC-3′ (SEQ ID NO:33).

Tumor inoculations: Female BALBIc homozygous (nu/nu) mice, 6-8 weeks ofage were maintained in a pathogen-free environment. Animals wereinoculated by subcutaneous injection with LCC6 (2×10⁶) cells.

Immunohistochemistry: Tumor samples were embedded in O.C.T. and snapfrozen in preparation for cryosectioning. Immunohistochemical detectionwas performed on 5-10 pm sections, using primary antibody againstPECAM-1 (R&D systems, BBA7, 1:1000-1:5000 of a 1 mg/mL stock solution)and anti-mouse IgG (whole molecule) peroxidase conjugate (A9044, Sigma).Staining was visualized using the cell and tissue staining kit (HRPIDABanti-mouse, CTS002) obtained from R&D systems (Minneapolis, Minn.).

Tumor volume and microvessel density determination: Tumor measurementswere performed using microcalipers. Tumor volumes were estimated usingthe formula π16×(larger diameter)×(small diameter)² weekly afterinitiation of treatment (Tomayko and Reynolds (1989) Cancer Chemother.Pharmacol. 24:148-154). The microvessel density was calculated as theaverage number of PECAM (CD-31)-stained microvessels within threemicroscopic fields containing the maximum number of discretemicrovessels, usually at the tumor periphery (Vermeulen et al. (1996)Eur. J. Cancer 32A:2474-2484).

Results: To investigate the effect of δS2 on tumor growth, thepT2caggs-δS2 expression construct, along with the pT2caggs parentalvector, were stably transfected into cells from the LCC6 breast tumorline. Transgene expression in the stable clones was confirmed by RT-PCR.A stable clone with δS2 transgene expression was expanded, and cellswere subsequently inoculated into the flanks of athymic nude mice.Compared to the tumors from the control pT2caggs transfected cells,tumors from the δS2 stable clone were significantly smaller (FIG. 14,p=0.04).

To determine whether there might be a correlation between tumor growthand vessel density, anti-CD 31 (PECAM) immunohistochemical staining wasperformed, and vessel density was determined as described above.Compared to tumors derived from LCC6 cells transfected with the parentalpT2caggs vector, tumors derived from cells transfected with δS2 showedlower vessel density (FIG. 15). These results indicate that slower tumorgrowth exhibited by δS2-transfected tumors correlates with lessangiogenic activity.

To determine the effect of δS2 on the growth of pre-established tumors,the Sleeping Beauty (SB) transposon-based system was used as anintratumoral gene transfer vector for breast tumor xenografts. Thissystem has been demonstrated to be an effective gene transfer vector forglioblastoma xenografts (Ohlfest et al., in press). To assess whetherSB-mediated gene transfer of δS2 would have a therapeutic effect onbreast tumor growth, tumors were first established in female nude miceby subcutaneous inoculation with LCC6 tumor cells. SB and δS2 DNAcassettes were then introduced into pre-established tumors that were 4-6mm diameter by injecting a solution containing SB and δS2 DNA:linearpolyethylenimine (PEI) complexes. As controls, DNA cassettes containingSB and the luciferase reporter gene (pT2-Luc) were introduced intotumors in another group of nude mice. Tumor size was measured weeklypost-injection. After 7 weeks, four of the five tumors injected with δS2and SB were smaller compared to the tumors injected with pT2-Luc and SB(FIGS. 16A and 16B).

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method for inhibiting angiogenesis in a vertebrate, said methodcomprising administering to said vertebrate an effective amount of acytoplasmically truncated Syndecan-2 polypeptide.
 2. The method of claim1, wherein said truncated Syndecan-2 polypeptide is a dominant negativeSyndecan-2 polypeptide.
 3. The method of claim 1, wherein said truncatedSyndecan-2 polypeptide comprises amino acids 1 to 193 of the sequenceset forth in SEQ ID NO:2.
 4. A method for inhibiting angiogenesis in avertebrate, said method comprising administering to said vertebrate aneffective amount of a nucleic acid comprising a sequence that encodes acytoplasmically truncated Syndecan-2 polypeptide.
 5. The method of claim4, wherein said construct is expressed in said vertebrate to producesaid truncated Syndecan-2 polypeptide.
 6. The method of claim 4, whereinsaid truncated Syndecan-2 polypeptide is a dominant negative Syndecan-2polypeptide.
 7. The method of claim 4, wherein said truncated Syndecan-2polypeptide comprises amino acids 1 to 193 of the sequence set forth inSEQ ID NO:2.
 8. A method for killing a tumor cell, said methodcomprising contacting said tumor cell with a cytoplasmically truncatedSyndecan-2 polypeptide.
 9. The method of claim 8, wherein saidcontacting comprises administering to said tumor cell a nucleic acidcomprising a sequence that encodes said cytoplasmically truncatedSyndecan-2 polypeptide.
 10. The method of claim 9, wherein saidconstruct is expressed in said tumor cell to produce said truncatedSyndecan-2 polypeptide.
 11. The method of claim 8, wherein saidtruncated Syndecan-2 polypeptide is a dominant negative Syndecan-2polypeptide.
 12. The method of claim 8, wherein said truncatedSyndecan-2 polypeptide comprises amino acids 1 to 193 of the sequenceset forth in SEQ ID NO:2.
 13. The method of claim 8, wherein said tumorcell is present in a breast tumor, a lung tumor, or a prostate tumor.14. The method of claim 13, further comprising monitoring the size ofsaid tumor.
 15. A method for inhibiting tumor growth, said methodcomprising contacting said tumor with a cytoplasmically truncatedSyndecan-2 polypeptide.
 16. The method of claim 15, wherein saidcontacting comprises administering to said tumor a nucleic acidcomprising a sequence that encodes said cytoplasmically truncatedSyndecan-2 polypeptide.
 17. The method of claim 16, wherein saidconstruct is expressed in said a cell of said tumor to produce saidtruncated Syndecan-2 polypeptide.
 18. The method of claim 15, whereinsaid truncated Syndecan-2 polypeptide is a dominant negative Syndecan-2polypeptide.
 19. The method of claim 15, wherein said truncatedSyndecan-2 polypeptide comprises amino acids 1 to 193 of the sequenceset forth in SEQ ID NO:2.
 20. The method of claim 15, wherein said tumorcell is present in a breast tumor, a lung tumor, or a prostate tumor.21. The method of claim 15, further comprising monitoring the size ofsaid tumor.